Several methods are available to assay for the presence or concentration of a predetermined analyte, like an ion, in a test sample. These materials include wet phase and dry phase colorimetric assays, and assays based on flame photometry, atomic absorption photometry, ion selective electrodes and multiple liquid phase partitioning. Recently, the ion selective electrode method of analysis has been more widely used, especially in regard to automated systems, as improvements in ion selective electrodes have developed. In particular, ion selective electrodes now have sufficient selectivity, sensitivity and operating lifetimes to be useful in automated systems.
One important aspect of an ion selective electrode is the use of particular compounds or compositions that preferentially or selectively complex with, and therefore isolate, a predetermined analyte, usually an ion, from a test sample. These compounds, known as ionophores, have the capability of selectively isolating a predetermined ion from its counterion and from other ions in the test sample, thereby causing a charge separation and a corresponding change in electrical conductivity in the phase containing the ionophore.
Ion selective electrodes therefore have been used to assay a test sample for the presence or concentration of a predetermined ion, either anionic or cationic, in solution. The prior art describes a variety of ion selective electrode types and structures to detect or measure a particular predetermined ion in solution. In general, devices that detect or measure the presence or concentration of a predetermined ion include a reference electrode and an electrode that responds preferentially or specifically to the predetermined ion in the test sample. When the reference electrode and the ion selective electrode each are immersed in solutions including differing concentrations of the predetermined ion, an electrical potential is generated in the electrochemical cell. This electrical potential is measured, and is correlated to the concentration of the predetermined analyte in the test sample.
In particular, when two solutions having unequal concentrations of the predetermined ion are separated by an electrically conductive membrane, an electromotive force (EMF) is generated. The EMF developed by the electrochemical cell is a function of the concentration, or the ionic activity, of the solutions on either side of the membrane. This phenomenon is expressed mathematically by the Nernst Equation (1): ##EQU1## wherein E is the EMF of the particular electrochemical cell, F is the Faraday constant, R is the gas constant, T is the temperature in .degree.K. (degrees Kelvin) and a.sub.1 and a.sub.2 are the activities of the predetermined ion in solution. The subscript 1 denotes the solution on one side of the membrane; the subscript 2 denotes the solution on the other side of the membrane The electrical charge of the predetermined ion is denoted by n.
The measurement is a differential potentiometric measurement of potential differences arising between the two identical electrochemical half-cells that are immersed in solutions of different activity and are separated by a salt bridge or a membrane. The two half-cells together comprise a concentration cell In the present case, the activity of one half-cell (a.sub.1) is fixed (reference), whereas the activity of the other half-cell (a.sub.2) (sample) is variable, such that the EMF of the concentration cell is defined from the Nernst equation (Eq. 1).
In such electrochemical concentration cells, the membrane can be a simple fritted glass barrier that allows a small, but measurable, degree of ion diffusion from one solution to the other solution on the opposite side of the membrane. Alternatively, a nonporous, electrically-nonconductive membrane, such as polyvinyl chloride, impregnated with an ionophore can be employed. In the absence of the ionophore, the membrane is an insulator and no EMF can be measured. When an ionophore is incorporated into the membrane, charged ions are bound to the membrane and a small, measurable current can be induced to flow. Such cells are ion selective because the ionophore preferentially or selectively binds to, or complexes with, the predetermined ion. Thus, the ionophore binds essentially only to the predetermined ion and any measurable EMF is due solely to the presence of the predetermined ion.
For example, it is known that certain antibiotics, such as valinomycin, have an effect on the electrical properties of phospholipid bilayer membranes (biological membranes). These antibiotics solubilize cations within the membrane in the form of mobile charged complexes, thereby providing a "carrier" mechanism whereby cations can cross the insulating hydrophobic interior of the membrane. These cation-antibiotic complexes have the sole purpose of carrying the charge of the complex through the hydrophobic membrane. In an ion selective electrode (ISE), the cation-antibiotic complexes generate a measurable voltage differential between the solutions on either side of the ISE membrane.
Therefore, a concentration cell for determining potassium ion concentration in a test sample results from using an ionophore, e.g. valinomycin, that is specific for potassium ion (K.sup.+). In the presence of potassium ions, valinomycin incorporated into a suitable membrane produces a concentration gradient across the membrane by binding and transporting the potassium ion, thus generating an electric potential across the membrane. A known, reference concentration of potassium ion contacts one side of the membrane and the test sample contacts the other. The resulting EMF is measured using external reference electrodes, and the measured EMF is used to calculate the unknown concentration of potassium ions in the test sample from equation (1). Because essentially only potassium ion binds to the valinomycin present in the membrane, the conductive path only appears for potassium ions. Therefore, the measured EMF is attributable solely to the potassium ion concentration gradient across the membrane. The actual current flowing across the membrane is so small that no significant quantity of potassium ion or counterion is transported through the membrane. Electrical neutrality of the membrane is maintained either by a reverse flow of hydrogen ions (protons), or by a parallel flow of hydroxyl ions.
This differential measurement technique has been used to measure the concentration or activity of constituents of biological fluids, such as hydrogen ion (H.sup.+), sodium (Na.sup.+), potassium (K.sup.+), calcium (Ca.sup.++) and chloride (Cl.sup.-). In addition, this technique often employs biosensors or enzyme electrodes that include a biological catalyst (e.g., immobilized enzymes, cells, or layers of tissues) coupled to an electrode and are sensitive to a product or cosubstrate of the biologically catalyzed reaction. Accordingly, the concentration of an enzyme or of a substrate can be determined using differential measurement techniques.
In the past, an ISE generally comprised an electrode body, usually a type of glass container containing a reference solution of known ion concentration in contact with a half-cell of known potential, generally written as Ag/AgCl/"XMCl"; and an ion selective glass membrane mounted in an aperture in the electrode body such that, when the electrode was immersed in the solution including an unknown concentration of the ion, the glass membrane contacted both the reference solution within the electrode body and the unknown solution An appropriate metal probe (Ag, silver) coated with a layer of an insoluble salt (AgCl, silver chloride) of the metal immersed in the contained reference solution ("XMCl", molar concentration of metal chloride) served as the contact and provided a reference potential for the electrode. The selectivity of the electrode for a particular ion was determined by the composition of the glass membrane or the components included in the glass membrane. Such electrodes are referred to as "barrel" electrodes, and are described in detail in U.S. Pat. Nos. 3,598,713 and 3,502,560.
More recently, the development of synthetic, polymeric membranes as substitutes for the glass membrane has increased the list of ions that can be assayed potentiometrically by an ion selective electrode method. The synthetic membranes generally comprise a polymeric binder or support impregnated with an ion selective ionophore and a solvent for the ionophore. Membranes of this type are custom-designed to preferentially or selectively sense a predetermined ion by a judicious selection of the ionophore, ionophore solvent, polymeric binder and other adjuvants incorporated into the polymeric binder. These synthetic membranes and "barrel" electrodes containing these membranes as substitutes for the glass membranes are described in detail in U.S. Pat. Nos. 3,562,129, 3,691,047, and 3,753,887. Other patents relating to ion selective electrodes include Yamaguchi, et al. U.S. Pat. No. 4,839,020, disclosing a gas sensor to assay for carbon dioxide; Burgess et al. U.S. Pat. No. 4,818,361, disclosing a combination electrode to measure pH and free carbon dioxide; Watkins-Pitchford U.S. Pat. No. 4,743,352; and Conover et al. U.S. Pat. No. 4,713,165.
The principal advantage of the ion selective "barrel" electrodes, in addition to their high specificity, is that the electrode can be used repeatedly for measuring the concentration of the same ion in different solutions. Accordingly, the assay of a large number of samples for a specific predetermined ion can be performed by automated devices. Presently therefore, many types of ion selective electrodes are available to measure the ion content of a liquid. These ion selective electrodes have limitations, however, including the requirement for membranes comprised of specially designed polymer matrices; utilization of ionophores that require pre-neutralization with base to improve membrane sensitivity and to reduce response time; the need for storage under well-controlled conditions; short useful lifetimes; and loss of sensitivity and reliability during storage. In addition, some ion selective electrodes require relatively large samples (i.e., 1 ml, or greater) for accurate operation and are made of glass. Therefore, the ion selective electrode is costly, fragile and cannot be incorporated into a device suitable for automatically processing samples of very small size. Another major shortcoming of some ion selective electrodes is that after the first use of the electrode to determine the ionic activity of an unconditioned fluid, such as a body fluid, the exact composition of the electrode membrane, either glass or polymeric, is unknown due to contamination by previously-assayed test samples. Therefore, assay results often are suspect.
As stated previously, an ion selective electrode (ISE) can be designed to assay a test sample for a predetermined cation or a predetermined anion. For example, Chapoteau et al., in U.S. Pat. No. 4,810,351, disclosed an ion selective electrode that assays a test sample for carbonate ions. The ISE can be used in an automated assay device, such as the TECHNICON RA-1000.RTM. random-access discrete analyzer system, available from Technicon Instruments Corp., Tarrytown, N.Y., to assay 240 or more samples per hour. As will be demonstrated more fully hereinafter, the method of the present invention is especially useful in an ion selective electrode-based assay of a test sample for carbonate ion. However, the method of the present invention can be used in the detection and measurement of any anion or cation that can be assayed by ISE techniques. Accordingly, the following discussion relating to the assay of a test sample, like a biological fluid, for carbonate ion is merely illustrative. By using a heavy metal-free composition that buffers the test sample within the appropriate pH range for the assay of interest, the method of the present invention can be used to assay for any of a variety of anions or cations by an ion selective electrode including a membrane having the appropriate ionophore.
Therefore, the detection or measurement of carbonate ion, that in turn is related to the total carbon dioxide content of a biological sample, like blood serum or plasma, is a clinically important assay. This assay is used in the diagnosis and treatment of several potentially-serious disorders associated with changes in the acid-based balance in the body. The normal pH of plasma is 7.4 and defines the ratio of bicarbonate ion (HCO.sub.3 --) to carbonic acid (H.sub.2 CO.sub.3) in the test sample by the Henderson-Hasselbalch equation. Any disturbance in blood pH is compensated by appropriate responses of the respiratory and renal systems. Hence, more than one analysis is required to determine acid-based status. One such assay is the analysis of the total carbon dioxide content of the blood. Carbon dioxide dissolved in blood is in equilibrium between the interior of red blood cells and the plasma and also within the plasma. Carbon dioxide is present as dissolved carbon dioxide (CO.sub.2), carbonic acid (H.sub.2 CO.sub.3), bicarbonate (HCO.sub.3.sup.--), carbonate (CO.sub.3.sup.-2) and carbonate bound to free amino groups of proteins (RNHCOO.sup.--). The total carbon dioxide concentration is defined as the sum of the concentrations of all forms of carbon dioxide that are present in the test sample.
In most assay methods for total carbon dioxide in blood serum or plasma, the biological fluid is added to an acidic reagent that converts bound carbon dioxide (HCO.sub.3.sup.--, CO.sub.3.sup.-2 and RNHCO.sub.2.sup.--) into free carbon dioxide (H.sub.2 CO.sub.3 and dissolved CO.sub.2). To determine total carbon dioxide, extraction methods, like dialysis, or equilibration methods measure the increase in pressure of gas at a fixed volume. In addition, potentiometric determination of the total carbon dioxide concentration has been performed using a carbonate-sensitive ion selective electrode. The ion selective electrode method requires fixing the pH of the test sample at a relatively high value, i.e., above about 8, by the addition of a buffered alkaline solution prior to testing, such as described in Herman and Rechnitz, Anal. Chem. Acta., 76, pp. 155-164 (1975).
Carbonate-sensitive ion selective electrodes are described in Wise, U.S. Pat. No. 3,723,281; Kim et al., U.S. Pat. No. 4,272,328; Meyerhoff et al. Anal. Chem. Acta, 141, pp. 57-64 (1982) and Simon et al., Anal. Chem., 54, pp. 423-429 (1982). A chloride-sensitive ion selective electrode is described in Oka et al , Anal. Chem., 53, pp. 588-593 (1981). Each above-cited patent or publication attempted to provide a carbonate-sensitive ISE that was accurate, had an acceptable operating life, and exhibited minimal drift. The prior art references also attempted to eliminate or reduce the effects of interferents, like gentisate or salicylate, that often are present in the test sample.
The previously-described disclosed ion selective membrane electrodes useful for determination of total carbon dioxide in biological fluids in automated analyzers exhibit interference from several sources, like fatty acids, keto acids, salicylate and heparin. However, judicious selection of membrane components has provided a membrane with superior performance characteristics, like a short conditioning time, a long lifetime in storage, a rapid and stable response, low drift, and significantly less susceptibility to interference. Such electrodes can analyze 240 samples or more per hour, and assay results correlate well with assays performed by a dialysis method for total carbon dioxide.
Chapoteau et al., in U.S. Pat. No. 4,810,351, disclosed this type of carbonate-sensitive ISE that can be used in automated assays for carbonate ion. Chapoteau et al. disclosed a flow-through ion selective electrode, wherein a test sample is diluted at a 1 to about a 15 dilution ratio of test sample to buffer solution The buffer solution has a pH greater than about 8.2 such that the test sample includes carbonate ions. Furthermore, the buffer solution includes a heavy metal ion complex, like a mercury(II) ethylenediaminetetraacetate complex, to reduce the effects of interferents, like salicylate, that often are present in the test sample. The ISE disclosed by Chapoteau et al. is capable of assaying over two hundred samples per hour.
The ion selective electrode method of assaying for total carbon dioxide disclosed by Chapoteau et al. should be contrasted to the prior art methods. The determination of total carbon dioxide in automated flow systems customarily involved acidification of the sample, followed by dialysis of the resulting carbon dioxide gas into a recipient stream. The quantity of carbon dioxide dialyzed is proportional to the total carbon dioxide content of the sample. The resulting change in the pH of the recipient stream is measured either colorimetrically or with a pH electrode. However, neither method was practical at the very fast sampling rates of more than about 200 samples per hour in comparison to the use of an ion selective electrode that performs the required assay without dialysis
Carbon dioxide reacts with water to form carbonic acid, that dissociates into bicarbonate ions and then into carbonate ions. The acid dissociation constants for these two dissociations is 6.37 and 10.25, respectively, at 25.degree. C. In a buffered medium, then, a suitable ion selective sensor for carbon dioxide can be responsive to either carbonate or bicarbonate ion. A bicarbonate sensor has been used, but its response times are excessively long (5 to 15 min). Carbonate-responsive devices therefore are more commonly used, even though the sensitivity of the carbonate-sensitive ISE is only about one-half that expected for a bicarbonate sensor. Furthermore, a carbonate-sensitive ISE requires a relatively high pH for the sample, that usually is achieved by adding an alkaline buffer solution to the sample prior to the assay.
Initial attempts to produce an ion selective electrode for a carbonate determination were plagued by poor selectivity in the presence of chloride, by poor analytical slopes in the range of physiological concentrations, and by failure to control pH. In addition, relatively unstable liquid membranes having slow response times were used, and assay results were complicated by occasional unexplained high analytical recoveries. Furthermore, both endogenous and other common components of serum, like free fatty acids, heparin, coumadin and salicylate, can interfere with the response of carbonate sensors.
The continuously reusable carbonate-sensitive ISE disclosed in U.S. Pat. No. 4,810,351 also is described in the publication by W. J. Scott, E. Chapoteau and A. Kumar, "Ion-Selective Membrane Electrode for Rapid Automated Determinations of Total Carbon Dioxide", in Clin. Chem., 3211, pp. 137-141 (1986). This electrode overcame many of the disadvantages of the prior art carbonate-sensitive and bicarbonate-sensitive ion selective electrodes. In addition, the ion-selective electrode could be arranged in sequence with sodium and potassium ion selective electrodes such that one test sample could be assayed for carbonate, sodium and potassium concentrations. The assays utilizing this carbonate-sensitive ISE are performed on a test sample diluted with a suitable buffer solution. The authors further stated that in addition to the components comprising the membrane of the ion selective electrode, the effects of anionic interferents present in the test sample also are further reduced by complexing the anionic interferents in solution, prior to the assay, with a suitable complexing reagent. Attempts to reduce the interfering effects of salicylate with ferric chloride, aluminum sulfate, caffeine, human serum albumin, or triazole were unsuccessful, as was an attempted carboxylation with peroxidase and an attempted oxidation with polyphenol oxidase or sodium hypochlorite. Another publication directed to an ion selective electrode assay method for carbonate ions is "Measurement of Total Carbon Dioxide Made at Low Range with an Ion Selective Electrode (TECHNICON RA-1000.RTM.)", W. J. Scott, E. Chapoteau and A. Kumar, Clin. Chem., 32(11), p. 2119-2120 (1986).
Therefore, investigators generally attempted to reduce the effects of interferents by improving the design of the ion selective electrode membrane. The only known prior art method of effectively reducing the interfering effects of anionic compounds, such as salicylate, gentisate, hypaque, heparin and coumadin, in a test sample is to dilute the test sample with a buffer solution including a metal complex. The addition of a metal complex to reduce the effects of an anionic interferent in an ion selective electrode-based assay is disclosed by Kumar, in U.S. Pat. No. 4,196,056.
Kumar disclosed including a heavy metal complex in the diluting buffer solution to reduce the interfering effects of iodide ion and bromide ion in the assay for chloride ion with an ion selective electrode. The heavy metal complex forms soluble complexes with the bromide and iodide ions, and therefore the bromide and iodide ions are not available to interact with the ion selective electrode. It should be understood that this is important because the ion selective electrode used to assay for chloride ion also is responsive to bromide and iodide ions; and, if detected, the bromide and iodide ion concentrations would produce a measured chloride ion concentration far in excess of the actual chloride ion concentration because of the logarithic measurement of the Nernst equation. The salicylate ion produces a similar response in the assay of a test sample for carbonate ion.
Although the method and composition disclosed by Kumar effectively reduce the effects of interfering anions, including iodide ions, bromide ions and other anions illustrated in the Scott et al. publication, the Kumar method and composition have the disadvantage of relying upon a heavy metal complex to reduce the effects of interferents. The metal complexes disclosed by Kumar include chelates of mercury, silver, lead, bismuth, copper and cadmium, and preferably include chelates of mercury(II). Many of these metals possess inherent toxicity, and therefore pose potential dangers to technicians that continually use buffers including the metal complexes Furthermore, considering the number of samples that are assayed (i.e. 240 or more per hour) and the dilution ratio of test sample to buffer (1 to at least 10), a relatively large volume of waste material is generated. According to the method of Kumar, this waste material includes a heavy metal, and therefore is difficult to dispose of safely and economically.
However, in accordance with an important feature of the present invention, a heavy metal-free composition is used to dilute and buffer the test sample. Surprisingly, the heavy metal-free composition effectively reduces the effects of interferents present in the test sample, while maintaining electrode sensitivity and maintaining a useful electrode lifetime. The composition and method of the present invention include a buffering compound, and preferably a borate compound, to dilute the test sample, to buffer the test sample at a suitable pH, and to reduce the effects of interferents. In addition to the buffering compound, the composition includes an alkalinity adjusting compound to provide a suitable pH for the particular assay of interest. For example, in the assay for carbonate ion, tetramethylammonium hydroxide and a borate compound provide a buffering composition having suitably high pH such that all of the carbon dioxide species in the test sample are converted to the carbonate ion. In the case of an assay for carbonate ion, the buffering composition also includes a small amount of bicarbonate ions to provide a small background amount of carbonate ions Optionally, the buffering composition includes a nonionic surface active agent to help improve ISE responses by enhancing the wash characteristics of the flowing stream. It should be understood that the metal-free composition includes a buffering compound, like a borate, to buffer the diluted test sample and to eliminate the effects of interferents.
If the ion selective electrode-based assay is for an analyte other than carbonate, then an alkalinity adjusting compound is selected to provide the appropriate pH for that particular analyte. In addition, the optional bicarbonate anion is eliminated, and, if desired, replaced by another suitable compound to provide a background concentration of the analyte of interest. Furthermore, for any analyte that is assayed between a pH of about 5 and about 11, a borate compound can serve as a buffer and to reduce the effects of an interferent. Outside of the pH range of from about 5 to about 11, the borate compound still is added to reduce the effects of an interferent, but a separate buffering compound should be added to maintain the diluted test sample at the desired pH.
Prior art electrodes have utilized a borate compound as a buffering agent. For example, Macur, in U.S. Pat. No. 3,957,613, disclosed a miniature probe for simultaneously sensing ions and gaseous partial pressures. The partial pressure sensor for gases includes a gas-permeable membrane enclosing a compartment filled with a borate buffer at pH 4.8 to 5.4. Butler, in U.S. Pat. No. 4,060,750, disclosed a thin film polarographic sensor that can utilize a borate buffer. Neither device is similar to the ion selective electrodes and method utilized in the present invention.
Accordingly, it has been found that the heavy metal-free composition of the present invention provides accurate and sensitive assays for a predetermined analyte in an ion selective electrode assay technique. The heavy metal-free composition can be used in automated assay devices wherein a single predetermined analyte in the test sample is assayed, or wherein several predetermined analytes are assayed in sequence by different ion selective electrodes. In addition, the heavy metal-free composition is safer to use, and waste disposal problems of the spent composition are overcome. Hence, in contrast to the prior art, new and unexpected results are achieved in the ion selective electrode-based assay of a predetermined analyte in a test sample, like blood plasma or serum, by utilizing a heavy metal-free composition as the buffering composition to dilute and buffer the test sample and to substantially reduce the effects of interferents often present in the test sample The heavy metal-free composition also maintains the selectivity of the ion selective electrode, and maintains the operating lifetime of the ion selective electrode. The heavy metal-free composition is especially useful in an ion selective electrode assay method for carbonate ion concentration in a test sample.